Prochlorococcus growth rates in the central equatorial ... - CiteSeerX

JOURNAL OF GEOPHYSICAL RESEARCH, VOL. 104, NO. C2, PAGES 3391-3399, FEBRUARY 15, 1999

Prochlorococcus growth rates in the central equatorial Pacific: An application of thef,,, approach Hongbin Liul and Michael R. Landry Department of Oceanography, School of Ocean and Earth Science and Technology, University of Hawaii at Manoa, Honolulu

Daniel Vaulot Station Biologique de Roscoff, Universitt Pierre et Marie CurieiCentre National de la Recherche ScientifiqueiInstitut National des Sciences de I’Univers, Roscoff Cedex, France

Lisa Campbell Department of Oceanography, Texas A&M University, College Station

Abstract. Minimum daily growth rates of Prochforococcus were estimated for the central equatorial Pacific (12%12”N, 14o”W) during El Nifio (February-March 1992) and normal upwelling (August-September 1992) conditions. Growth rate estimates were based on the percentages of cells in the S and G2 division phases at dawn (-0700 LT) and dusk (-1800 LT) as approximate values for f,i” and f,,,, respectively. During both environmental conditions, depth-integrated growth rates of Prochlorococcus were higher in oligotrophic waters at the northern end of the transect (0.36-0.52 d-l) and decreased to a minimum at the equator. The lowest growth rates were found at the equator during El Nirio and at 2”N during normal upwelling, where a large biomass of buoyant diatoms had accumulated in the vicinity of a convergent front. Prochlorococcus growth rate reached a high of 0.64 dK’ at 1”s and maintained a moderate rate (0.36-0.49 d-‘) throughout the southern end of the transect. An inverse relationship was found between the contribution of Prochlorococcus to the total primary production and nitrate concentration as well as total primary production measured by the 14C method. While Prochlorococcus is a dominant primary producer in tropical and subtropical open-ocean ecosystems generally, it is relatively more important in oligotrophic waters than in the nutrient rich equatorial upwelling zone.

Despite its relatively recent discovery in the mid-1980s [Chisholm et al., 19881 the prokaryotic picoplankter Prochlorococcus is now recognized as a major component of photosynthetic biomass throughout tropical and subtropical regions of the open oceans. Interest in understanding the factors that influence its rates .of growth and contributions to primary production is thus considerable. Pigment labeling [e.g., Gieskes and Kraay, 19891, flow cytometric sorting [e.g., Li, 19941, and dilution [La&y et al., 1995a, b] techniques have all been applied with varying degrees of success to these growth and production rate measurements; however, cell cycle analysis is particularly well suited for this organism because of its bimodal DNA distribution and synchronous cell division [Vaulot et al., 19951. The cell cycle approach derives growth rate estimates from the fractions and durations of S (DNA synthesis) and G, (DNA replication complete but not yet divided) phases of the ambient population over a die1 cycle and thus avoids incubation artifacts [Vaulot et al., 1995; Liu et al., 19971. However, the

approach requires frequent sampling over a 24 hour cycle, which limits its use to situations where a station will be occupied for a day or more and where the time demands of other sampling activities do not conflict. Such conditions are rarely met in major oceanographic research programs with multiple investigators and scientific agendas. Such was the case for survey cruises of the U.S. Joint Global Ocean Flux Study (JGOFS) Equatorial Pacific (EqPac) Program in 1992 which sampled along a transect of stations from 12”s to 12”N, 14O”W during both El Nifio and normal upwelling conditions. Routine dawn and dusk water column samples during these cruises were not sufficient for standard cell cycle analysis, but they lend themselves to a modified f,,,,, approach [Vuulot, 19921 which uses the percentages of S + G, cells at dawn and dusk to approximate values forf,,, andf,,,,,, respectively. In the present study we use this approach to examine variations in Prochlorococcus growth rates and contributions to total primary production across the environmental gradient from oligotrophic to high-nutrient equatorial waters and during El Niiio and normal upwelling conditions.

‘Now at Marine Science Institute, University of Texas at Austin, Port Aransas.

2. Materials and Methods

Copyright 1999 by the American Geophysical Union.

2.1.

Paper number 1998JC900011. 0148-0227/98/19985C900011$09.00

There are two main ways to compute in situ growth rates from cell cycle analysis: (1) from the total number of cells in a

1. Introduction

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Growth Rate Estimates From Cell Cycle Analyses

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LIU ET AL.: PROCHLOROCOCCUS GROWTH RATE IN THE EQUATORIAL PACIFIC

cell cycle terminal event (duration = td) over the 24 hour period and (2) from the daily maximum fraction of cells in the terminal event. The first category is illustrated by the Carpenter and Chang [1988] model, which is based on the derivation by McDuff and Chisholm [1982]. The most recent version of this model using S + G, phases as the terminal event was presented by Liu et al. [1997] as .?J /+W’) = & .i0

In [ 1 +

fs+df)l dt

(1)

where p,, is the average specific growth rate over the photoperiod t,, (usually 24 hours),f,+,?(t) is the fraction of cells in the S + G, phases of cell division at time point t in the 24 hour sampling period, and tStG, is the duration of S + G, computed as twice the time between the peak fraction of cells in S and the peak fraction of cells in G, (see notation section) [Carpenter and Chang, 19881. This equation is based on the assumption that all cells within the population have the same t, (in our case, t,,,); thus it is not accurate if part of the population becomes dark-arrested in the terminal event. Moreover, since tS+G is determined from the time interval between observed peaks of cells in the S and G, phases the quality of this estimate is strongly dependent on the frequency of population sampling. In contrast to the time-integrated p,, calculation, the “f”,,__ approach” does not require knowledge of t,. McDuff and Chisholm [1982] concluded that when the terminal phase t,, is long compared to the cytokinesis, then P/,,~,,> = 1 it,) In ( 1 + fmd

(2)

wherein fmZ,, is the maximal fraction of cells in the terminal phase of the cell cycle (see notation section). Growth rate estimates from (2) give a lower bound to the daily division rate because f,,;,, decreases when phasing becomes less tight [see McDuff and Chisholm, 1982, Figure 1; Antiu et al., 19901. Vaulot [1992] developed a more general form of (2) for phased cell division, including the possibility that slow growing cells spend more than 1 day in the terminal phase: (3) where f,,,,, and f,,,i, are the daily maximal and minimal fractions of cells in the terminal phase of the cell cycle (e.g., S + Gz) (see notation section). When t, < t,,, f,,,,, is equal to zero, and (3) reduces to (2). In the present study we used the fraction of Prochlorococcus cells in the S + G, phases from the samples taken at 0700 ( f,,7,,,I) and 1800 ( f, 8,,o) LT as proxies for f,,i, and fnlaX, respectively. Prochlorococcus has been shown to have a tightly phased cell cycle beginning with DNA replication in late afternoon followed by cell division in the evening [Vaulot et al., 1995; Partensky et al., 1996; Liu et al., 1997, 19981. The exact timing for the time of maximumf,,,, is generally only known within an hour or two because of coarse sampling over the die1 cycle. Nonetheless, it is apparent that cells near the surface usually begin DNA synthesis and cell division later than the cells deeper in the euphotic zone (Figure 1). Interregional comparisons suggest that Prochlorococcus enters S and G, phases slightly later in oligotrophic regions, such as Station ALOHA in the subtropical North Pacific (Figure 1). Because the time of maximum fStci, varies within the water column and among different regions, depth-profile samples from -1800 LT will likely

50

0

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24

Local Time of Day Figure 1. Depth profiles of the time at which the maximum fraction of Prochlorococcus cells in S + Gz phases were observed during diel sampling in different oceanic regions: Central Equatorial Pacific (C-Eqpac), including data from o”, 15o”W (Flux dans lbuest du Pacifique Equatorial (FLUPAC), October 1994) [Liu et al., 19971, o”, 14O”W (EqPac TT008 and TT012, April and October 1992) [Vaulot et al., 19951, and 5”, 15O”W (Oligotrophie en Pacifique (OLIPAC), November 1994) [Vaulot and Marie, this issue]; Station ALOHA, U.S. Joint Global Ocean Flux Study (JGOFS) Hawaiian Ocean Time Series (HOT) Station ALOHA (22”45’N, 158”W), during HOT 55 (July 1994) 64 (July 1995) 65 (August 1995) and 70 (March 1996); Arabian Sea, 5 stations in the Arabian Sea, ranging from oligotrophic open ocean to mesotrophic coastal waters, during U.S. JGOFS Arabian Sea process cruises TN050 (August 1995) and TN054 (December 1995) [Liu et al., 19981; and Western Equatorial Pacific (W-EqPac), including the oligotrophic western equatorial Pacific (00, 166”E) during French JGOFS FLUPAC cruise in October 1994 [Liu et al., 19971. The solid vertical line shows the time of 1800 LT.

capture only part of the population at maximum S + G,, leading to underestimates in the growth rates at other depths and the depth-integrated average rate. 2.2. Sample and Data Analysis During EqPac cruises TTOO7 (El Niiio conditions; FebruaryMarch 1992) and TTOll (normal upwelling conditions: August-September 1992) seawater samples were collected at 10 m depth intervals to 120 m and at 150 and 200 m from early morning (between 0600 and 0800 LT) and late afternoon (between 1730 and 1830 LT) hydrocasts at each of I5 stations along a transect from 12”N to 12”S, 14O”W. These samples were previously analyzed by dual-beam flow cytometry for abundances and distributions of picoplankton populations

LIU ET AL.: PROCHLOROCOCCUS GROWTH RATE IN THE EQUATORIAL PACIFIC Growth Rate (d-‘) 0

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Figure 2. Comparison of (a) Pan,,, pfmin,max, and pp and (b) ~~,s,,, ~~~~~~~ 181,0r and CL, of Prochlorococcus from a die1 sampling collected from the central equatorial Pacific (15O’W) during the FLUPAC cruise in October 1994 [Liu, 19951. The I*,,, pfmax, pfmin,maxr P,=]~~“, and ~~0700,,800 terms are defined in the notation section.

(DNA staining with Hoechst 33342 after Monger and Landry [1993]; see Landry et al. [1996] for detailed descriptions of sample collection and analytical protocols). For the present study we reanalyzed the listmode files by first projecting the data for the Prochlorococcus population to a set of singleparameter histograms and converting the histograms of logblue (DNA) fluorescence to ASCII format using CYTOPC software [Vaulot, 19891. The integrated log-blue fluorescence data were then converted to a linear scale using the following equation XL,, = A X 10B

X

X,,,/total channels

(4)

where XLi, and X,,, are the signal channels in linear and log scale and A and B are coefficients for the logarithmic decadal scale (e.g., A. = 1 and B = 3 corresponds to a 3 decade logarithmic scale). For the photomultiplier tube (PMT) amplifier of log-blue fluorescence in our flow cytometer, A and B were 0.18 and 3.16, respectively. The rebuilt, linear blue fluorescence histograms were analyzed with MCYCLE (Phoenix Flow Systems, San Diego, California) to compute the percentage of Prochlorococcus cells in the S and G, phases. Growth rates of Prochlorococcus, /+uf800 and ~~070,,,800 were computed using (2) and (3), respectively. Growth rates were integrated throughout the depth range of positive growth using the formula described by Vaulot et al. [1995] that weights division rates by cell concentrations at each depth: N,&z)e pL(‘) dzl

*,“I = ln [ I

I

N,m&z)

dzl

(5)

where z is the water depth, p(z) is the growth rate estimate at depth z, and N,8,Jo(z) is the cell concentration of Prochlorococcus at depth z at -1800 LT. Depth-integrated rates of daily carbon production of Pro-

chlorococcus were estimated from cell concentrations in dusk samples and cell division rates according to

p=

cc,,,

N,8,,,,(z)[eP'z'

- 11 dz

(6)

where Ccc,, is the intracellular carbon content of Prochlorococcus, estimated as 53 fg C cell-’ [Campbell et al., 19941. In (5) and (6) we used cell concentrations from the 1800 LT sampling as approximations of the minimum cell concentrations just before division. Primary production estimates from the 14C assimilation method and nutrient data were obtained from JGOFS data system available at http://wwwl.whoi.edu/ eqpac.html [see also Barber et al., 1996; Archer et al., 19961.

3. Results and Discussion 3.1. Comparison of Coniputational Methods To assess differences in growth rate estimates among com-

putational methods, we compared I.+ I+,,,,,, Pfmin,max, I.+ 8oo

and ~fo7,~0,,80,, for Prochlorococcus using depth profiles collected over a die1 cycle in the central equatorial Pacific in October 1994 [Liu, 1995; Liu et al., 19971. The pfrnax and P~,,,~“,,,~~ values were calculated from (2) and (3), respectively, using the observed maximum fs+G2 during die1 sampling. The results show that CL,, estimates exceed j+,,,, by -0.1 dK’ throughout the upper 80 m of the water column (Figure 2). It was anticipated that +,,,, would underestimate /.L~ because of imperfect synchrony in the natural dividing population [Vaulot, 19921 and that the discrepancy would be exaggerated if limited sampling failed to hit the real peak of cells in the S + G, phases. On the other hand, inaccuracies in the estimate of t S+GZ due to sampling frequency and high “background”fs+G2 during the nondivision period could also have caused /.L,, to

LIU ET AL.: PROCHLOROCOCCUS GROWTH RATE IN THE EQUATORIAL PACIFIC

3394

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Figure 3. Examples of depth profiles of Prochlorococcus growth rate calculated with p, , xoo and ~,,,,,,,,, , x,,,, approaches from three stations on the 14O”W transect during (top) El Niiio and (bottom) normal upwelling conditions, the TTOO7 and TTOll cruises, respectively.

overestimate the true growth rates of Prochlorococcus [Liu et al., 19971. In comparing the w~,,,;,~ method with its P~,~,~~, proxy for this data set the latter calculation underestimated F~,,,~,~ by as much as 37% at the surface and below 60 m (Figure 2b). The differences between the growth rate estimates from (2) and (3) were generally smaller, implying that only a minor fraction of the cells were still dividing in the dawn samples. In the EqPac data set we observed few, if any, G, cells in 1800 LT samples from the upper layers of oligotrophic regions, indicating that cell division had not yet occurred. In mesotrophic equatorial waters, however, a significant fraction of the near-surface cells were already in the G, phase at 1800 LT. This difference in timing may explain the inconsistent pattern of Prochlorococcus abundance between morning and evening samplings noted in the equatorial Pacific [Landry et al., 19961. Prochlorococcus were less abundant in the evening (-1800 LT) than in the morning (-0700 LT) in oligotrophic regions at both ends of the transect, and the reverse was true near the equator. Light-dependent dephasing of the cell cycle may be a strategy to avoid damage from high irradiance, including UV penetration [I’uulot et al., 199.51. The depth of the surface layer that shows the delay of S and G, phases extends deeper in oligotrophic regions as the result of deeper light penetration in

the clearer water (Figure l), and we have noticed that the S phase starts later in oligotrophic compared to mesotrophic waters [Liu et al., 19971. In addition to environmental conditions the difference in timing of the maximum S + G, among geographic locations may also be related to strain variations. Laboratory experiments have shown that different strains react differently in photosynthetic performance and growth kinetics to light intensity and quality [Purtensky et al., 1993; Moore et al., 19951. Experiments have also demonstrated that Synechococcus, a close relative of Prochlorococcus, entered the S phase earlier when phosphorus was added to a P-limited natural population [I/dot et al., 19961. In addition, die1 samples from on-deck incubations in oligotrophic waters of the Arabian Sea revealed that Prochlorococcus cells enter S phase earlier in shipboard bottle incubations compared to parallel conductivity-temperature-depth (CTD) sampling (H. Liu, unpublished data, 1997). This implies that the vertical mixing within the surface mixed layer may play a role in regulating the cell cycle timing of Prochlorococcus. If the Prochlorococcus cell cycle follows the block point model proposed by Spudich and Sager [1980], regional and seasonal variations in day length should also affect dephasing. Because of the complex dephasing of the Prochlorococcus cell cycle, it is impossible for a single time point sample to catch the maximum of S + Gz phases at all

LIU ET AL.: PROCHLOROCOCCUS GROWTH RATE IN THE EQUATORIAL PACIFIC

I____----._r __._ _.7~--_----__r -12

_

-10

-8

-6

-4

-i

+--T_.-. -12 -10 -8

-6

-4

-2

3305

8

,5 0

Latitude (North) Figure 4. Contour plots of I*~, xo,~ for Prochlorococcus along the 14O”W cross-equatorial transect from 12”N to 12’S during (top) February-March and (bottom) August-September 1992.

depths at a given station. Repeated sampling around the expected peak time would enhance the probability of catching the real peak offs + (;, at different depths. 3.2. Seasonal and Spatial Variations in Prochlorococcus Growth Rates Growth rate estimates increased slightly with depth to a deep maximum at stations from 7” to 12”N (Figure 3). The subsurface peak was better developed during the upwelling cruise around 40-100 m. From 5”s to S’N the maximum growth rates of Prochlorococcus occurred in the lo-30 m depth range. Growth rates decreased sharply with depth below the maximum, except at 0” and 1”s during El Nifio, where the decrease below 30 m was more gradual. At the southern end of the transect (12”s) the depth profile of the Prochlorococcus growth rate was similar to that at the northern end, except that the depth of the maximum growth rate was shallower (-50 m) during normal upwelling. One common feature on both cruises was an apparent secondary maximum in Prochlorococcus growth rate between 90 and 120 m. At stations near the equator we observed two well-separated populations of Prochlorococcus around 100 m. The dim (low-chlorophyll) population had a lower growth rate and disappeared below 100 m, whereas the bright (high-chlorophyll) population grew faster and extended deeper (Figure 3). These and similar observations [e.g., Campbell and Vaulot, 1993; Reckermann and Veldhuis, 19971 suggest that multiple Prochlorococcus populations in the deep chlorophyll maximum layer may be a common feature of tropical and subtropical oceans. Seasonal and spatial differences in Prochlorococcus growth rates were observed between cruises. Contour plots reveal higher rate estimates in the surface water around the equator

and during normal upwelling conditions (Figure 4). Maximum growth rates in the oligotrophic waters generally occurred at a deeper depth, with higher rates during El Niiio conditions. Depth-averaged growth rates of Prochlorococcus along the 14O”W transect displayed similar patterns for both cruises (Figure 5). Maximum depth-integrated rates were observed at IS, in what is typically the Equatorial Divergence. The maximum growth rate at 1”s matched the highest measured rate of primary production during the 1992 El Niiio-Southern Oscillation (ENSO), but not later during normal upwelling conditions, when larger phytoplankton increased in relative importance [Bid&are and Ondrusek, 19963. Prochlorococcus growth rates were usually high at the northern end of the transect (7”-12”N), where population abundances were highest [Landry et al., 19961, and they decreased toward the equator as the distribution of Prochlorococcus shifted closer to the surface. Unlike the El Nifio cruise, when the minimum in Prochlorococcus growth rate occurred at the equator, the lowest growth during the normal upwelling cruise was in the vicinity of a convergent front at 2”N where a large biomass of buoyant diatoms had accumulated [Yoder et al., 1994; Barber et al., 19941. As noted by Landy et al. [ 19961, Prochlorococcus abundance was also markedly depressed at that front. The percentage of S and Gz cells in the 0700 LT samplings were low in the surface waters but increased with depth. This trend may reflect an increasing proportion of cells unable to finish their S or G, phases before dawn, especially in deeper waters below the 0.1% light level. For both seasonal transects the percentages of Prochlorococcus cells in the S + G2 phases in the dawn samples ( f,j70,,) were -5%-10% at most stations and as high as 20% at a few stations. These percentages were

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GROWTH RATE IN THE EQUATORIAL PACIFIC

Table 1.

Contribution of Prochlorococcus to Total Primary Production in Different Regions Along the 14o”W CrossEquatorial Transect Normal Upwelling

El Niiio Condition

7’-12”N

5”N-5”s

12”s

7”- 12”N

5”N-5”s

12”s

NO, concentration in surface water, pmol kg-’ Prochlorococcus growth rate, d&’

0.20

4.58

0.42

0.11

2.38

(1.26

0.46

0.43

0.49

0.49

0.44

0.46

Total 14C primary production, mg C m ’ d-’

296

1117

313

336

6%

403

Prochlorococcus contribution, %

79

16

59

53

21

52

The percentage of Prochlorococcus contribution has been justified by multiplying 14C primary production values by 2 (D. M. Karl), and Prochlorococcus production estimates by two thirds (see discussion in text). All rates are integrated to the depth of 0.1% light level.

fraction of cells in terminal phase at 1800 LT. fraction of cells in S and G, phases. average growth rate over the photoperiod t,, hK’. estimate of growth rate from f,,, (equation (2)), h-‘. estimate of growth rate from f,,,,, and f,;,, (equation (3)), h _I. estimate of growth rate from S, x,jo (equation (2)), h- ‘. estimate of growth rate from fo7,,,, and f, 8,10 (equation (3)), hK’. depth-integrated growth rate, d- ‘. cell concentration at depth z at 1800 LT. Acknowledgments. We thank J. Kirshtein and J. Constantinou for sample collection and initial analysis and H. A. Nolla for technical assistance in data analysis. Comments from two reviewers improved the manuscript. This research was supported by National Science Foundation grants OCE 90-22117 (MRL), 93-11246 (MRL and LC), and 94-17071 (LC). DV was supported by EU contract MAST CT950016 (MEDEA). Contributions 4699 from the School of Ocean and Earth Science and Technology, University of Hawaii at Manoa, and 481 from the U.S. JGOFS Program.

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LIU ET AL.: PROCHLOROCOCCUS

GROWTH RATE IN THE EQUATORIAL PACIFIC

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(Received January 9, 1998; revised August 10, 1998; accepted August 31, 1998.)